A linear fatty acid or linear ester rhein derivative having a hydroxyl group, and a preparation method and application thereof

Abstract

The present invention belongs to the field of medicinal chemistry, and particularly relates to a linear fatty acid or linear ester rhein derivative having a hydroxyl group, a preparation method and an application thereof. The rhein derivative described in the present invention has the general formula (I) or a pharmaceutically acceptable salt thereof: #imgabs0# wherein the R1 group is a linear fatty acid or a linear ester. The rhein derivative provided by the present invention is a compound based on natural rhein. By modifying the carboxyl group of the rhein structure and introducing a linear fatty acid and a linear ester with a hydroxyl group, the anti-inflammatory activity of such compounds is improved. Compared with rhein, the rhein derivative of the present invention has significantly reduced cytotoxicity to macrophages and has a better anti-inflammatory effect, and can be used for preparing drugs for treating inflammation.

Description

Technical Field

[0001] The present invention relates to rhein derivatives, and specifically to a linear fatty acid or linear ester rhein derivative with a hydroxyl group, and its preparation method and application. Background Art

[0002] In view of the emergence of current anti-inflammatory drug resistance and the increase in related diseases, the mechanism of inflammation has also received extensive attention. Clarifying the anti-inflammatory mechanism and seeking drugs with high efficiency and low toxicity and side effects have become hotspots in medical research. At present, the main anti-inflammatory drugs in China are mainly steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs, and are widely used in clinical practice. With the extensive application of anti-inflammatory drugs, there have also been some cases of cross-infection caused by the abuse of anti-inflammatory drugs, and long-term use will also cause a series of side effects. Therefore, the emergence of new anti-inflammatory drugs is urgently needed.

[0003] There are many types of natural compounds in China, mainly including: terpenoids, saponins, flavonoids, anthraquinones, alkaloids, etc. Finding effective monomers from them and obtaining anti-inflammatory drugs with high activity and low toxicity through modification and transformation is the current main research direction. Rhein, as a plant antibiotic, has activities such as anti-tumor, anti-virus, anti-diabetic nephropathy, antibacterial and anti-inflammatory. Therefore, rhein has once become a research hotspot.

[0004] Although rhein has a wide range of pharmacological effects, its clinical application is greatly limited due to defects such as poor water solubility and low bioavailability. Therefore, using the active ingredient of the natural product rhein as the parent compound and performing structural modification according to chemical molecular principles to design semi-synthetic compounds with high activity and low toxicity and side effects, so as to discover new molecular entities that may be applied clinically is an important means of current new drug development. Summary of the Invention

[0005] In order to overcome the above problems existing in the current prior art, the present invention has studied a linear fatty acid or linear ester rhein derivative with a hydroxyl group, and its preparation method and application.

[0006] The present invention first provides a linear fatty acid or linear ester rhein derivative with a hydroxyl group, having the general formula (I) or a pharmaceutically acceptable salt thereof:

[0007]

[0008] In the formula, the R1 group is a linear fatty acid or linear ester.

[0009] The rhein derivative of the present invention is a compound obtained by chemically modifying the isolated and purified rhein with a linear fatty acid or linear ester with a hydroxyl group.

[0010] The second object of the present invention is to provide a method for preparing the above-mentioned rhein derivatives, and the steps include: mixing rhein, a straight-chain fatty acid or straight-chain ester with a hydroxyl group, 4-dimethylaminopyridine, and dicyclohexylcarbodiimide, adding solvents dimethyl sulfoxide and tetrahydrofuran, carrying out an esterification reaction, dialyzing and drying the reaction product, and then purifying and drying to obtain the rhein derivative.

[0011] The preparation reaction of the rhein derivative of the present invention is shown by the following reaction formula:

[0012]

[0013] Preferably, the straight-chain fatty acid or straight-chain ester containing a hydroxyl group can be selected from pentadecanohydroxy acid, 10-hydroxydecanoic acid, 12-hydroxystearic acid, ricinoleic acid, or diolein.

[0014] Preferably, the molar ratio of rhein, the straight-chain fatty acid or straight-chain ester with a hydroxyl group, 4-dimethylaminopyridine, and dicyclohexylcarbodiimide is 1:1:0.1:1.

[0015] Preferably, the temperature range of the esterification reaction is 45°C - 65°C, and the time of the esterification reaction is 24h - 48h.

[0016] Preferably, the dialysis is first carried out through a sodium bicarbonate solution. After dialysis for 12h - 24h, then dialysis with pure water for 24 - 48h.

[0017] Preferably, the purification is carried out by column chromatography, and the volume ratio of ethyl acetate to petroleum ether in the chromatography solution is 2:3 - 1:1.

[0018] Another object of the present invention is to provide the application of the above-mentioned rhein derivative in the preparation of anti-inflammatory drugs.

[0019] Among them, the rhein derivatives prepared by the present invention include:

[0020]

[0021] After performing cytotoxicity and anti-inflammatory experiments, it was found that compared with rhein at the same concentration, the cytotoxicity to macrophages was significantly reduced, and it had a better anti-inflammatory effect than rhein.

[0022] The present invention also provides an anti-inflammatory drug containing a rhein derivative, which is an anti-inflammatory drug composed of the above-mentioned rhein derivative or a pharmaceutically acceptable salt thereof as an active ingredient, alone or in combination with one or more pharmaceutically acceptable carriers or excipients.

[0023] The drug can be used in the form of injection, tablet, pill, capsule, suspension or emulsion, and its administration routes can be oral, intravenous or intramuscular injection, topical application, etc.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] The linear fatty acid or linear ester rhein derivative with a hydroxyl group provided by the present invention has lower cytotoxicity to macrophages than rhein at the same concentration, and both have better anti-inflammatory effects. They have an inhibitory effect on the release of NO from LPS-activated Raw264.7 cells and inhibit the production of ROS in LPS-damaged macrophages. Even better effects can be achieved. The rhein derivatives synthesized by the present invention can be used to prepare anti-inflammatory drugs. Description of the Drawings

[0026] Figure 1 Cytotoxicity analysis diagram of rhein derivative R-15HA on macrophages Raw264.7 ( Figure 1 A); anti-inflammatory effect diagram on LPS-damaged macrophages ( Figure 1 B-1D), Figures B-D are respectively the level change diagrams of inflammatory factors TNF-α, IL-6 and IL-1β; effect diagram on the NO content in the supernatant of LPS-damaged macrophages Raw264.7 ( Figure 1 E).

[0027] Figure 2 Cytotoxicity analysis diagram of rhein derivative R-10HA on macrophages Raw264.7 ( Figure 2 A); anti-inflammatory effect diagram on LPS-damaged macrophages ( Figure 2 B-2D), Figures B-D are respectively the level change diagrams of inflammatory factors TNF-α, IL-6 and IL-1β; effect diagram on the NO content in the supernatant of LPS-damaged macrophages Raw264.7 ( Figure 2 E).

[0028] Figure 3 Cytotoxicity analysis diagram of rhein derivative R-12HA on macrophages Raw264.7 ( Figure 3 A); anti-inflammatory effect diagram on LPS-damaged macrophages ( Figure 3 B-3D), Figures B-D are respectively the level change diagrams of inflammatory factors TNF-α, IL-6 and IL-1β; effect diagram on the NO content in the supernatant of LPS-damaged macrophages Raw264.7 ( Figure 3 E).

[0029] Figure 4 Cytotoxicity analysis diagram of rhein derivative R-RA on macrophages Raw264.7 ( Figure 4A); Anti-inflammatory effect diagram of macrophages damaged by LPS ( Figure 4 B-4D), Figures B-D are the level change diagrams of inflammatory factors TNF-α, IL-6, and IL-1β respectively; Effect diagram of the influence on the NO content in the supernatant of macrophages Raw264.7 damaged by LPS ( Figure 4 E).

[0030] Figure 5 Cytotoxicity analysis diagram of rhein derivative R-GD on macrophages Raw264.7 ( Figure 5 A); Anti-inflammatory effect diagram of macrophages damaged by LPS ( Figure 5 B-5D), Figures B-D are the level change diagrams of inflammatory factors TNF-α, IL-6, and IL-1β respectively; Effect diagram of the influence on the NO content in the supernatant of macrophages Raw264.7 damaged by LPS ( Figure 5 E).

[0031] Figure 6 Cytotoxicity analysis diagram of rhein derivative R-2HA on macrophages Raw264.7 ( Figure 6 A); Anti-inflammatory effect diagram of macrophages damaged by LPS ( Figure 6 B-6D), Figures B-D are the level change diagrams of inflammatory factors TNF-α, IL-6, and IL-1β respectively; Effect diagram of the influence on the NO content in the supernatant of macrophages Raw264.7 damaged by LPS ( Figure 6 E).

[0032] Figure 7 Cytotoxicity analysis diagram of rhein derivative R-16HA on macrophages Raw264.7 ( Figure 7 A); Anti-inflammatory effect diagram of macrophages damaged by LPS ( Figure 7 B-7D), Figures B-D are the level change diagrams of inflammatory factors TNF-α, IL-6, and IL-1β respectively; Effect diagram of the influence on the NO content in the supernatant of macrophages Raw264.7 damaged by LPS ( Figure 7 E).

[0033] Figure 8 Cytotoxicity analysis diagram of rhein derivative R-5HA on macrophages Raw264.7 ( Figure 8 A); Anti-inflammatory effect diagram of macrophages damaged by LPS ( Figure 8 B-8D), Figures B-D are the level change diagrams of inflammatory factors TNF-α, IL-6, and IL-1β respectively; Effect diagram of the influence on the NO content in the supernatant of macrophages Raw264.7 damaged by LPS ( Figure 8 E). Specific implementation manners

[0034] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0035] Unless otherwise specified, the test methods used in the embodiments of the present invention are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels; the equipment used, unless otherwise specified, are all conventional experimental equipment.

[0036] Example 1 Preparation of Rhein Derivative R-15HA

[0037] (1) Synthesis and Purification of Rhein Derivative R-15HA

[0038] Weigh 1 g of rhein, 0.91 g of pentadecanedioic acid, 0.043 g of 4-dimethylaminopyridine, and 0.72 g of dicyclohexylcarbodiimide into a two-necked flask, add 20 ml of dimethyl sulfoxide (DMSO) and 10 ml of tetrahydrofuran (THF), and stir and react at 50 °C for 24 h.

[0039] Transfer the solution obtained above into a dialysis bag (500D), dialyze with saturated sodium bicarbonate and pure water for 24 h each, and freeze-dry the obtained semi-solid product for 48 h. Subsequently, the solid powder is further purified by a chromatography column (chromatography solution: ethyl acetate / petroleum ether = 2; 3), the purified extract is obtained, rotary evaporated under reduced pressure and concentrated, and finally dried to obtain the end product.

[0040] (2) Product Identification

[0041] The product obtained in step (1) is confirmed by FTIR, NMR and Ms data.

[0042] Result analysis: Rhein derivative, yellow powder solid, molecular weight: 524.62, molecular formula: C 30 H 36 O3.

[0043] 1 HNMR(300MHz,DMSO)δ: 11.89(d,1H), 8.09(d,0H), 7.89–7.68(m,1H), 7.41(dd,0H), 4.39(q,1H), 4.02(dq,0H), 2.37–2.15(m,1H), 1.55–1.47(m,1H), 1.43–1.12(m,12H);

[0044] IR(KBr), 3300, 1675, 2800;

[0045] FAB-MS m / z(%) : 526 [M+H] - 。

[0046] This yellow powder solid is simply referred to as R-15HA. After identification, its structural formula is shown as follows:

[0047]

[0048] Preparation of Rhein Derivative R-10HA in Example 2

[0049] (1) Synthesis and Purification of Rhein Derivative R-10HA

[0050] Weigh 1 g of rhein, 0.66 g of 10-hydroxydecanoic acid, 0.043 g of 4-dimethylaminopyridine, and 0.72 g of dicyclohexylcarbodiimide into a two-necked flask, add 20 ml of dimethyl sulfoxide (DMSO) and 10 ml of tetrahydrofuran (THF), and stir and react at 50 °C for 24 h.

[0051] Transfer the solution obtained above into a dialysis bag (500D), dialyze with saturated sodium bicarbonate and pure water for 24 h each, and subject the resulting semi-solid product to freeze-drying for 48 h. Subsequently, further purify the solid powder through a chromatography column (chromatography solution: ethyl acetate / petroleum ether = 2:3), purify and extract to obtain a solution, rotary evaporate under reduced pressure for concentration, and dry to obtain the final product.

[0052] (2) Product Identification

[0053] Confirm the product obtained in step (1) by FTIR, NMR, and Ms data.

[0054] Result Analysis: Rhein derivative, yellow powder solid, molecular weight: 454.16, molecular formula: C 25 H 26 O8.

[0055] 1 H NMR (600 MHz, DMSO) δ 11.91 (d, 1H), 8.04–7.44 (m, 1H), 4.34 (t, 1H), 2.38–1.92 (m, 3H), 1.75 (p, 1H), 1.60–0.98 (m, 20H);

[0056] IR (KBr), 3300, 1675, 2800;

[0057] FAB-MS m / z(%) : 454.2 [M+H]-.

[0058] This yellow powder solid is simply referred to as R-10HA. After identification, its structural formula is as shown below:

[0059]

[0060] Preparation of Rhein Derivative R-12HA in Example 3

[0061] (1) Synthesis and Purification of Rhein Derivative R-12HA

[0062] Weigh 1 g of rhein, 1.06 g of 12-hydroxy stearic acid, 0.043 g of 4-dimethylaminopyridine, and 0.72 g of dicyclohexylcarbodiimide into a two-necked flask, add 20 ml of dimethyl sulfoxide (DMSO) and 10 ml of tetrahydrofuran (THF), and stir and react at 50 °C for 24 h.

[0063] Transfer the solution obtained above into a dialysis bag (500D), dialyze with saturated sodium bicarbonate and pure water for 24 h each, and subject the obtained semi-solid product to freeze-drying for 48 h. Subsequently, further purify the solid powder through a chromatography column (chromatography solution: ethyl acetate / petroleum ether = 2:3), purify and extract to obtain a solution, rotary evaporate under reduced pressure and concentrate, and dry to obtain the final product.

[0064] (2) Product Identification

[0065] Confirm the product obtained in step (1) through FTIR, NMR, and Ms data.

[0066] Result Analysis: Rhein derivative, yellow powder solid, molecular weight: 566.29, molecular formula: C 33 H 42 O8.

[0067] 1 HNMR(300MHz,DMSO)δ11.91(s,2H),8.18(d,1H),7.99–7.61(m,2H),7.53–7.19(m,2H),5.57(d,1H),4.24–3.93(m,1H),1.87–1.06(m,32H),0.95–0.61(m,3H);

[0068] IR(KBr), 3300, 1675, 2800;

[0069] FAB-MSm / z(%):568.1[M+H]-.

[0070] This yellow powder solid is simply referred to as R-12HA. After identification, its structural formula is as shown below:

[0071]

[0072] Preparation of Rhein Derivative R-RA in Example 4

[0073] (1) Synthesis and Purification of Rhein Derivative R-RA

[0074] Weigh 1 g of rhein, 1.05 g of ricinoleic acid, 0.043 g of 4-dimethylaminopyridine, and 0.72 g of dicyclohexylcarbodiimide into a two-necked flask, add 20 ml of dimethyl sulfoxide (DMSO) and 10 ml of tetrahydrofuran (THF), and stir and react at 50 °C for 24 h.

[0075] Transfer the solution obtained above into a dialysis bag (500D), dialyze with saturated sodium bicarbonate and pure water for 24 h each, and subject the obtained semi-solid product to freeze-drying for 48 h. Subsequently, further purify the solid powder through a chromatography column (chromatography solution: ethyl acetate / petroleum ether = 2:3), purify and extract to obtain a solution, concentrate it under reduced pressure by rotary evaporation, and dry it to obtain the final product.

[0076] (2) Product Identification

[0077] Confirm the product obtained in step (1) by FTIR, NMR, and Ms data.

[0078] Result Analysis: Rhein derivative, yellow powder solid, molecular weight: 564.27, molecular formula: C 33 H 40 O8.

[0079] 1 1H NMR (400 MHz, DMSO) δ 11.91 (d, 1H), 8.46–8.07 (m, 1H), 7.97–7.70 (m, 2H), 7.51–7.23 (m, 2H), 5.53–5.04 (m, 1H), 4.28–3.95 (m, 1H), 2.11–1.60 (m, 9H), 1.53–0.92 (m, 19H), 0.89–0.67 (m, 3H);

[0080] IR (KBr), 3300, 1675, 2800;

[0081] FAB-MS m / z (%) : 565.3 [M + H]-.

[0082] This yellow powder solid is simply called R-RA. After identification, its structural formula is shown as follows:

[0083]

[0084] Preparation of Rhein Derivative R-GD in Example 5

[0085] (1) Synthesis and Purification of Rhein Derivative R-GD

[0086] Weigh 1 g of rhein, 2.18 g of diolein, 0.043 g of 4-dimethylaminopyridine, and 0.72 g of dicyclohexylcarbodiimide into a two-necked flask, add 20 ml of dimethyl sulfoxide (DMSO) and 10 ml of tetrahydrofuran (THF), and stir and react at 50 °C for 24 h.

[0087] Transfer the solution obtained above into a dialysis bag (500D), dialyze with saturated sodium bicarbonate and pure water for 24 h each, and freeze-dry the obtained semi-solid product for 48 h. Subsequently, further purify the solid powder through a chromatography column (chromatography solution: ethyl acetate / petroleum ether = 2:3), purify and extract to obtain a solution, concentrate it by rotary evaporation under reduced pressure, and dry it to obtain the final product.

[0088] (2) Product Identification

[0089] Confirm the product obtained in step (1) by FTIR, NMR, and Ms data.

[0090] Result analysis: Rhein derivative, yellow powder solid, molecular weight: 886.56, molecular formula: C 54 H 78 O 10 .

[0091] 1 HNMR(600 MHz, DMSO) δ 11.91 (d, 1H), 8.12 (d, 1H), 8.06–7.64 (m, 2H), 7.66–6.63 (m, 1H), 5.27 (d, 1H), 4.39 (q, 2H), 4.04 (q, 1H), 2.36–1.98 (m, 3H), 1.66–1.03 (m, 47H), 0.94–0.52 (m, 6H);

[0092] IR (KBr), 3300, 1675, 2800;

[0093] FAB-MS m / z (%) : 886 [M+H]-.

[0094] This yellow powder solid is simply called R-GD. After identification, its structural formula is shown as follows:

[0095]

[0096] Example 6 Preparation of Rhein Derivative R-2HA

[0097] (1) Synthesis and Purification of Rhein Derivative R-2HA

[0098] Weigh 1 g of rhein, 0.96 g of 2-hydroxyhexadecanoic acid, 0.043 g of 4-dimethylaminopyridine, and 0.72 g of dicyclohexylcarbodiimide into a two-necked flask, add 20 ml of dimethyl sulfoxide (DMSO) and 10 ml of tetrahydrofuran (THF), and stir and react at 50 °C for 24 h.

[0099] Transfer the solution obtained above into a dialysis bag (500D), dialyze with saturated sodium bicarbonate and pure water for 24 h each, and subject the obtained semi-solid product to freeze-drying for 48 h. Subsequently, the solid powder is further purified by a chromatography column (chromatography solution: ethyl acetate / petroleum ether = 2:3), the purified extract is obtained, rotary evaporated and concentrated under reduced pressure, and dried to obtain the final product.

[0100] (2) Product identification

[0101] Confirm the product obtained in step (1) by FTIR, NMR, and Ms data.

[0102] Result analysis: Rhein derivative, yellow powder solid, molecular weight: 538.26, molecular formula: C 31 H 38 O8.

[0103] 1 HNMR (400 MHz, DMSO) δ 11.93 (s, 1H), 8.44–7.98 (m, 1H), 7.94–7.54 (m, 1H), 5.55–5.08 (m, 1H), 4.22–3.85 (m, 1H), 2.04–1.49 (m, 2H), 1.54–1.05 (m, 29H), 0.85 (q, 3H);

[0104] IR (KBr), 3300, 1675, 2800;

[0105] FAB-MS m / z (%) : 538.3 [M + H]-.

[0106] This yellow powder solid is simply called R-2HA. After identification, its structural formula is shown as follows:

[0107]

[0108] Example 7 Preparation of Rhein derivative R-16HA

[0109] (1) Synthesis and purification of Rhein derivative R-16HA

[0110] Weigh 1 g of rhein, 0.96 g of 16-hydroxyhexadecanoic acid, 0.043 g of 4-dimethylaminopyridine, and 0.72 g of dicyclohexylcarbodiimide into a two-necked flask, add 20 ml of dimethyl sulfoxide (DMSO) and 10 ml of tetrahydrofuran (THF), and stir and react at 50 °C for 24 h.

[0111] Transfer the solution obtained above into a dialysis bag (500D), dialyze with saturated sodium bicarbonate and pure water for 24 h each, and freeze-dry the obtained semi-solid product for 48 h. Subsequently, the solid powder is further purified by a chromatography column (chromatography solution: ethyl acetate / petroleum ether = 2:3), the purified extract is obtained, rotary evaporated under reduced pressure and concentrated, and dried to obtain the final product.

[0112] (2) Product identification

[0113] Confirm the product obtained in step (1) by FTIR, NMR, and Ms data.

[0114] Result analysis: Rhein derivative, yellow powder solid, molecular weight: 538.26, molecular formula: C 31 H 38 O8.

[0115] 1 HNMR (300 MHz, DMSO) δ 11.92 (s, 1H), 8.00–7.52 (m, 1H), 7.52–7.05 (m, 1H), 3.15–2.66 (m, 1H), 2.34–1.88 (m, 4H), 1.25 (d, 28H), 1.04–0.68 (m, 3H);

[0116] IR (KBr), 3300, 1675, 2800;

[0117] FAB-MS m / z (%) : 538.3 [M + H]-.

[0118] This yellow powder solid is simply called R-16HA. After identification, its structural formula is shown as follows:

[0119]

[0120] Example 8 Preparation of Rhein derivative R-5HA

[0121] (1) Synthesis and purification of rhein derivative R-5HA

[0122] Weigh 1 g of rhein, 0.42 g of (S)-2-hydroxyvaleric acid, 0.043 g of 4-dimethylaminopyridine, and 0.72 g of dicyclohexylcarbodiimide into a two-necked flask, add 20 ml of dimethyl sulfoxide (DMSO) and 10 ml of tetrahydrofuran (THF), and stir and react at 50 °C for 24 h.

[0123] Transfer the solution obtained above into a dialysis bag (500D), dialyze with saturated sodium bicarbonate and pure water for 24 h each, and freeze-dry the obtained semi-solid product for 48 h. Subsequently, further purify the solid powder through a chromatography column (chromatography solution: ethyl acetate / petroleum ether = 2:3), purify and extract to obtain a solution, rotary evaporate and concentrate under reduced pressure, and dry to obtain the final product.

[0124] (2) Product identification

[0125] Confirm the product obtained in step (1) by FTIR, NMR, and Ms data.

[0126] Result analysis: Rhein derivative, yellow powder solid, molecular weight: 384.08, molecular formula: C 20 H 16 O8.

[0127] 1 1H NMR (300 MHz, DMSO) δ 11.90 (s, 2H), 8.91 (t, 1H), 8.27–7.58 (m, 3H), 7.56–7.32 (m, 1H), 2.26–1.77 (m, 1H), 1.55–1.02 (m, 9H), 0.90 (dt, 1H);

[0128] IR (KBr), 3300, 1675, 2800;

[0129] FAB-MS m / z (%) : 385.4 [M+H]-.

[0130] This yellow powder solid is simply called R-5HA. After identification, its structural formula is shown as follows:

[0131]

[0132] Example 9 Toxicity and anti-inflammatory effect of rhein derivative

[0133] (1) Cytotoxicity of rhein derivative to macrophage Raw264.7

[0134] Select the rhein derivatives prepared in Examples 1-8 and the natural product rhein to conduct cytotoxicity experiments on macrophages (Raw264.7).

[0135] RAW264.7 cells were cultured in 24-well plates and grown to a density of about 60%. The drugs were diluted in DMEM to a final concentration of 5, 10, 20, 40, 80, 160 μM, and then incubated with the drugs for 24 hours. CCK-8 was then added and incubated for 2 hours, and the absorbance at 450 nm was measured to calculate the cell survival rate.

[0136] Results Analysis

[0137] like Figure 1-8 As shown in the A figures, the toxicity of rhein gradually increases with the increase of concentration; and the toxicity of rhein is changed after modification, and different grafted derivatives have different toxicities to RAW264.7. Among them, R-15HA and R-GD have a cell survival rate of about 90% at 160μM, and the toxicity is significantly lower than that of rhein, and there is a significant difference with rhein at the same concentration; the toxicity of the grafted derivatives R-10HA, R-12HA, and R-RA is lower than that of rhein, while the toxicity of R-2HA, R-16HA, and R-5HA is higher.

[0138] (2) Anti-inflammatory effects of rhein derivatives on LPS-damaged macrophages Raw264.7

[0139] In order to evaluate the anti-inflammatory effect of rhein and its grafted derivatives, the anti-inflammatory level of Raw264.7 damaged by LPS was detected. The anti-inflammatory effect of synthetic rhein derivatives and natural product rhein on LPS damaged macrophages (Raw264.7) was analyzed.

[0140] The QPCR method (parameters are shown in Table 1) was used. According to the toxicity results of step (1), the concentration of rhein and its grafted derivatives was selected to be 10 μM. Raw264.7 cells were incubated with drugs for 3 h and stimulated with 1 μg / mL LPS for 6 h. The RNA of Raw246.7 cells after treatment was then extracted, reverse transcribed, and qPCR was used to measure the expression levels of IL-1β, IL-6, and TNF-α to detect changes in the levels of inflammatory factors.

[0141] Table 1

[0142]

[0143] Results Analysis

[0144] like Figure 1-8As shown in each of the B-D diagrams in [reference], the detection of the levels of inflammatory factors in macrophages damaged by LPS found that after the action of the drug at 10 μM, rhein and graft derivatives both had obvious inhibitory effects on inflammation; among the graft derivatives R-15HA and R-5HA modified and transformed, their expression effects on IL-6 and IL-1β were significantly better than those of rhein, showing significant differences compared with rhein. R-10HA, R-12HA, R-2HA, R-RA, and R-GD had better effects compared with rhein, while the effect of R-16HA was not significantly different. Therefore, compared with rhein, the graft derivatives had better anti-inflammatory effects.

[0145] (3) Effect of Rhein Derivatives on the Content of NO in the Supernatant of LPS-Damaged Macrophage Raw264.7

[0146] NO produced by cells is easily oxidized to NO2-, and under acidic conditions, NO2 in the solution - can undergo a diazotization reaction with Griess reagent to form an orange-red substance. In this experiment, the changes in the content of NO in the supernatant of macrophages stimulated by LPS with rhein and graft derivatives were detected.

[0147] According to the toxicity results in step (1), the concentrations of rhein and graft derivatives were selected as 10 μM, and Raw264.7 cells were incubated with the drugs for 2 h, stimulated with 1 μg / mL LPS for 18 h, and then the supernatant (50 μl) was taken. Then, NO detection solution A (50 μL) and NO detection solution B (50 μL) were added, and each well was repeated 3 times. The reaction was carried out in the dark at room temperature for 10 min, and the absorbance was measured at a wavelength of 540 nm.

[0148] Result Analysis

[0149] The index results of NO are as Figure 1-8 shown in each of the E diagrams in [reference]. Under the stimulation of 1 μg / mL LPS, it can significantly induce the release of NO from Raw264.7 cells. Rhein has an inhibitory effect on the release of NO from LPS-activated Raw264.7 cells. The derivatives R-15HA, R-10HA, R-GD, R-16HA, R-5HA, and R-RA modified and transformed can significantly inhibit the release of NO, showing significant differences compared with rhein. While R-2HA and R-12HA have effects similar to those of rhein, both can play a role in inhibiting NO. Therefore, by comparison, it is found that the effects of rhein derivatives have been improved.

[0150] Obviously, the specific embodiments described above only further elaborate on the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above is only a specific example of the present invention and is not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A linear fatty acid or linear ester rhein derivative having a hydroxyl group, characterized in that, The structural formula of the rhein derivative is any one of the following formulas:

2. A method for preparing the rhein derivative according to claim 1, characterized in that, It includes mixing rhein, a straight-chain fatty acid or straight-chain ester containing a hydroxyl group, 4-dimethylaminopyridine, and dicyclohexylcarbodiimide, adding the solvents dimethyl sulfoxide and tetrahydrofuran, carrying out an esterification reaction, dialyzing and drying the reaction product, and then purifying and drying it to obtain the rhein derivative; the straight-chain fatty acid or straight-chain ester containing a hydroxyl group is selected from pentadecanolide, 10-hydroxydecanoic acid, 12-hydroxystearic acid, ricinoleic acid or diolein.

3. The preparation method of the rhein derivative according to claim 2, characterized in that, The molar ratio of the rhein, the straight-chain fatty acid or straight-chain ester containing a hydroxyl group, 4-dimethylaminopyridine, and dicyclohexylcarbodiimide is 1:1:0.1:

1.

4. The preparation method of the rhein derivative according to claim 2, wherein, The temperature of the esterification reaction is 50 °C, and the time of the esterification reaction is 24 h.

5. Use of the rhein derivative according to claim 1 in the preparation of an anti-inflammatory drug.

6. A rhein derivative anti-inflammatory drug, characterized in that, It is composed of the rhein derivative according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient, alone or in combination with one or more pharmaceutically acceptable carriers or excipients.

7. The drug according to claim 6, characterized in that, The drug can be an injection, an oral preparation, a spray, or a cream.

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